Method for inducing targeted meiotic recombinations

Abstract

The present invention relates to a fusion protein comprising a Cas9 domain and a Spo11 domain, as well as the use of this protein to induce targeted meiotic recombinations in a eukaryotic cell.

We claim: 
     
         1 . A method for inducing targeted meiotic recombination(s) in a plant cell comprising:
 introducing into a plant cell:   a) a fusion protein comprising a Cas9 protein lacking nuclease activity and a Spo11 protein, or a nucleic acid encoding said fusion protein; and   b) one or more guide RNAs or one or more nucleic acids encoding said guide RNAs, said guide RNAs comprising an RNA structure for binding to the Cas9 protein of the fusion protein and a sequence complementary to a targeted chromosomal region; and   inducing said cell to enter meiotic prophase I,   thereby inducing meiotic recombination(s) at the targeted chromosomal region.   
     
     
         2 . The method according to  claim 1 , wherein the fusion protein further comprises a nuclear localization signal sequence. 
     
     
         3 . The method according to  claim 1 , wherein the nucleic acid encoding said fusion protein is operably linked to a constitutive, inducible or meiosis-specific promoter. 
     
     
         4 . The method according to  claim 1 , further comprising introducing one or more additional guide RNAs targeting one or more other chromosomal regions, or nucleic acids encoding said additional guide RNAs. 
     
     
         5 . A method for generating variants of a non-human eukaryotic organism comprising introducing into a cell of said non-human eukaryotic organism:
 a) a fusion protein comprising a Cas9 protein lacking nuclease activity and a Spo11 protein, or a nucleic acid encoding said fusion protein; and   b) one or more guide RNAs, or one or more nucleic acids encoding said guide RNAs, said guide RNAs comprising an RNA structure for binding to the Cas9 protein and a sequence complementary to a targeted chromosomal region;
 inducing said cell to enter meiotic prophase I; 
 obtaining a cell or cells having recombination(s) at the targeted chromosomal region(s); and 
 generating a variant of the organism from said recombinant cell, 
   wherein the non-human eukaryotic organism is a plant.   
     
     
         6 . A method for identifying or locating genetic information encoding a characteristic of interest in a eukaryotic cell genome comprising:
 introducing into the eukaryotic cell:   a) a fusion protein comprising a Cas9 protein lacking nuclease activity and a Spo11 protein, or a nucleic acid encoding said fusion protein; and   b) one or more guide RNAs, or one or more nucleic acids encoding said guide RNAs, said guide RNAs comprising an RNA structure for binding to the Cas9 protein and a sequence complementary to a targeted chromosomal region;   inducing said cell to enter meiotic prophase I;   obtaining a cell or cells having recombination(s) at the targeted chromosomal region(s); and   analyzing genotypes and phenotypes of the recombinant cells in order to identify or to locate the genetic information encoding the characteristic of interest,   wherein the eukaryotic cell is a plant cell.   
     
     
         7 . The method according to  claim 6 , wherein the characteristic of interest is a quantitative trait of interest (QTL). 
     
     
         8 . A fusion protein comprising a Cas9 protein lacking nuclease activity and a Spo11 protein. 
     
     
         9 . A nucleic acid encoding the fusion protein according to  claim 8 . 
     
     
         10 . An expression cassette or a vector comprising the nucleic acid according to  claim 9 . 
     
     
         11 . The vector according to  claim 10 , said vector being a plasmid comprising:
 a bacterial or eukaryotic origin of replication,   an expression cassette comprising a nucleic acid encoding the fusion protein comprising a Cas9 protein lacking nuclease activity and a Spo11 protein under the control of an expression promoter,   one or more selection markers, and/or   one or more sequences allowing targeted insertion of the vector, the expression cassette or the nucleic acid into the genome of a host cell.   
     
     
         12 . A host cell comprising the fusion protein according to  claim 8 . 
     
     
         13 . The host cell according to  claim 12 , wherein said host cell is a eukaryotic cell. 
     
     
         14 . The host cell according to  claim 13 , wherein said eukaryotic cell is a yeast cell. 
     
     
         15 . The host cell according to  claim 13 , wherein said eukaryotic cell is a plant cell.